DESCRIPTION The goal of the proposed project is to investigate the molecular basis of cell type specific transcription of the fibronectin gene in normal and fibrotic liver. It is proposed to use rat liver to isolate hepatocytes, sinusoidal endothelial cells, lipocytes, and Kupffer cells. The liver fibrosis model proposed for examination of fibronectin gene expression in injury is ligation of the biliary duct in the rat. The region of the fibronectin promoter proposed for investigation includes a CRE and CCAAT motif. These studies will test the hypothesis that FN transcriptional regulation differs among the subpopulations of cells present in the liver in the context of injury. The specific aims include: 1) In vivo and cell culture assays to investigate the different liver cells for DNA binding proteins that interact with the CRE and CCAAT sites of the fibronectin promoter. Liver sections will be probed with antibodies specific for CREB, ATF-2, CP-1 and NF-1. Nuclear extracts from sinusoidal endothelial cells and hepatocytes will be used in DNA mobility shift assays with the fibronectin CRE and CCAAT sites to determine binding specificity (competition with normal and mutant oligonucleotides) and to identify transcription factors (competition, supershift, immunodepletion). DNase I footprinting will precisely identify sequences involved in factor binding. RNA blots will assess expression of the relevant transcription factor mRNAs in sinusoidal endothelial cells and hepatocytes. 2) Gene transfer and biochemical assays to compare fibronectin transcription in primary cultures of sinusoidal endothelial cells and hepatocytes. A nested set of fibronectin promoter-reporter genes will be used in cotransfections along with another reporter gene which is not cell-type specific. In vitro transcription studies will confirm transfection results and identify sites relevant to cell-type specific expression. For these studies, the wild type and mutant fibronectin promoters will control activity of transcription templates which direct synthesis of G-free transcripts. 3) Transfection and in vitro transcription to compare regulated fibronectin gene transcription in sinusoidal endothelial cells and hepatocytes for interaction between the CRE and CCAAT sites and the effects of signaling molecules. These studies will involve the use of spacing mutants, which alter the distance between the CRE and CCAAT motifs, and site mutants, which contain altered sequences within these motifs. Transcriptional activity of reporter genes will also be studied to determine the influence of regulatory molecules which affect DNA-protein interactions at the CRE/CCAAT site (cAMP, serum) or which promote fibroproliferative reactions (TGF-( and cytokines). 4) To investigate the functional relationship of promoter configuration/activity and the pattern of alternative splicing in the EIIIA region. This will involve transfection experiments with alpha globin/fibronectin minigenes which contain the EIIIA exon and expression will be directed by various promoters, including the fibronectin wild type, the CRE-CCAAT spacing mutants, the CRE and CCAAT site mutants, a liver specific promoter and 2 general promoters. Reporter transcripts will be assessed for the presence or absence of the EIIIA exon by RT-PCR.